For total protein extraction, hPSCs were lysed with RIPA buffer (Sigma-Aldrich) containing protease inhibitor cocktail (Roche) and phosphatase inhibitors cocktail 2 and 3 (Sigma-Aldrich). For histone extraction we used the Histone extraction protocol for western blot from Abcam (Cambridge; UK) web page. Cell lysates were separated by molecular weight using SDS-polyacrylamide gels and transferred to polyvinylidene fluoride (PVDF) membranes. Protein was detected using the Odyssey Infrared Imaging System (Li-cor Biosciences; Lincoln, NE, USA). To detect KDM5A and NUP98-KDM5A was used the α-KDM5A antibody (ab70892, Abcam). Also used α-NUP98 (ab50610, Abcam), α-HIF1A (610959, BD Bioscience; San Jose, CA, USA), α-γ-H2AX (#9718, Cell signalling; Danvers, MA, USA). An α-β-Actin (A5441, Sigma-Aldrich) was used as a loading control for total protein extractions and α-H3 (ab1791, Abcam) was used as a loading control in histone extractions. Western blotting was carried out using standard procedures. Quantification of band intensity and normalization was carried out using ImageJ (NHI, Bethesda, Maryland, USA, https://imagej.nih.gov/ij ).
α β actin
Manufactured by Merck Group
Sourced in United States
α-β-actin is a cytoskeletal protein that is found in all eukaryotic cells. It plays a crucial role in various cellular processes, including cell motility, structure, and signaling. The protein is composed of two isoforms, α-actin and β-actin, which are highly conserved across different species.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor cocktail, by Roche (5 mentions)
α gapdh, by Merck Group (4 mentions)
α flag, by Merck Group (4 mentions)
Chemidoc imaging system, by Bio-Rad (3 mentions)
α gfp, by Merck Group (2 mentions)
Chemidoc xr, by Bio-Rad (2 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (2 mentions)
αcdc73, by Fortis Life Sciences (2 mentions)
αpaf1, by Fortis Life Sciences (2 mentions)
αprmt5, by Merck Group (2 mentions)
αsym10, by Merck Group (2 mentions)
αh2bub, by Merck Group (2 mentions)
αh3k4me3, by Abcam (2 mentions)
αh3k79me2, by Abcam (2 mentions)
αh4r3me2s, by Abcam (2 mentions)
αr me2s, by Cell Signaling Technology (2 mentions)
α phospho erk1 2, by Cell Signaling Technology (2 mentions)
α phospho akt s473, by Cell Signaling Technology (2 mentions)
α phospho akt t308, by Cell Signaling Technology (2 mentions)
Monoclonal α flag m2 antibody, by Merck Group (2 mentions)
36 protocols using α β actin
1
Histone and Total Protein Extraction Protocols
2
Myosin IC Isoform A Antibody Characterization
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The myosin IC-isoform A antibody is a mouse monoclonal antibody that was raised against the myosin IC isoform A specific N-terminal peptide and recognizes exclusively myosin IC isoform A10 (link). The total myosin IC antibody recognizes a region in the tail domain that is common to all myosin IC isoforms (Santa Cruz Biotechnology, Dallas, TX). Other antibodies used: α-β-actin (Sigma-Aldrich, St Louis, MO); α-GFP (EMD Millipore; Billerica, MA); α-caveolin-1 (Cell Signaling Technologies, Danvers, MA); α-MMP1 (EMD Millipore; Billerica, MA), α-MMP9 (EMD Millipore; Billerica, MA); peroxidase-conjugated as well as Texas Red-conjugated secondary anti-mouse or anti-rabbit antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA).
3
Generation of CRISPR Knockout Cell Lines
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To generate the HEK-293SFΔTBK1 knockout pool, an sgRNA was designed using CHOPCHOP (v.2)49 (link) to target the first exon of the TBK1 gene (NCBI accession:NG_046906.1). sgRNA for the HEK-293SFΔDDX6, HEK-293SFΔSMG9, and HEK-293SFΔCARM1 knockout pools were randomly selected from corresponding Brunello library guides for that gene21 (link). This sgRNA was then cloned into LentiCRISPR.V2 (Addgene #52961) using standard techniques and verified by Sanger sequencing. LentiCRISPR.V2 was a gift from Feng Zhang50 (link). Lentiviral vectors produced using this construct were used to infect HEK-293SF at an MOI of 10. Following selection with 2 µg/mL Puromycin for 48 h, CRISPR editing efficiency was assessed using the TIDE webtool (v2.0.1)51 (link). Primers used to generate PCR amplicons for this analysis are listed in Supplemental S8 . Cells were then incubated for a further 10 days to allow knockout phenotypes to manifest and recover the drop in cell viability arising from DNA cleavage. TBK1 knockout was further verified by Western blot using mouse αTBK1 (Santa Cruz, sc9085) at a 1/200 dilution. αβ-Actin (Sigma A1978) at a 1/1,000 dilution was used as a loading control.
4
Analyzing Protein Expression in Cells
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Cells were washed in ice-cold PBS and then lysed in SDS sample buffer, supplemented with proteases inhibitors, as reported before. Total lysates were separated by SDS-PAGE and then transferred to nitrocellulose membrane ∅ 0.2 μm (GE Healthcare, WhatmanTM). Western blot analyses were performed according to standard procedures, using the following antibodies: αHMGA1 [13 (link)]; α-β-actin (A2066, Sigma); αFOXM1 (A301-533A-M, Bethyl Laboratories; D3F2B Cell Signaling Technology); αGFP (kind gift of L. Collavin, LNCIB, Trieste).
5
Western Blot Analysis of Histone Modifications
6
MG132 and Doxycycline Reagents Protocol
7
Cortical Neuron Protein Expression Analysis
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Cortical neurons were plated at a density of 1 × 106 cells/well on poly-L-lysine and laminin-coated pretreated six wells plates. Cells were collected with a scrapper into a homogenization buffer (250 mM Sucrose, 20 mM Hepes, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, Complete protease inhibitor cocktail mini-EDTA free, (Roche, Basel, Switzerland) and adjusted to pH 7.4). The samples were homogenized by sonication, and aliquots of 30 μg protein (Bradford assay) were separated by SDS-PAGE and transferred to nitrocellulose membranes. Primary antibodies used were α-ORAI1 (1:200) rabbit polyclonal, α-ORAI2 (1:200) rabbit polyclonal, α-STIM2 (1:500) rabbit polyclonal, α-TRPC1 (1:500) rabbit polyclonal, α-TRPC4 (1:500) rabbit polyclonal, (all from Alomone Labs, Jerusalem, Israel) and α−βActin (1:5000) mouse monoclonal (Sigma-Aldrich, St. Louis, MO, USA).
8
Western Blot Analysis of Cellular Proteins
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Protein lysates were prepared as detailed previously (Ahmed et al., 2019 ). Briefly, cells were resuspended in RIPA buffer containing Protease Inhibitor cocktail (Roche, Basel, Switzerland) on ice. The mixture was then centrifuged at 15 000 r.p.m. for 20 min at 4 °C. The supernatant was collected, and following protein concentration determination, lysates were separated by SDS/PAGE, transferred onto 0.2‐µm PVDF membranes (Bio‐Rad, Hercules, CA, USA) and blocked in superblock blocking buffer (Thermo Fisher Scientific, Waltham, MA, USA). Membranes were incubated with primary antibodies [α‐HMGA2 (CST 5269; 1 : 1000; RRID:AB_10694917), α‐TOP2A (TopoGEN TG2011‐1; 1 : 2000; RRID:AB_1934304), α‐TOP2B (Ab109524; 1 : 2000; RRID:AB_10859793), α‐β‐actin (Sigma A2228; 1 : 5000; RRID:AB_476697)] overnight at 4 °C, followed by incubation with secondary antibodies [Polyclonal goat anti‐mouse (Dako, P0447, Carpinteria, CA, USA; RRID:AB_2617137) and polyclonal goat anti‐rabbit (Dako, P0448; RRID:AB_2617138)] at room temperature for 1 h. Immunoreactivity was developed by chemiluminescent HRP substrate (Millipore, Singapore) in a luminescence imager (LAS4000; Fujifilm, Pittsburgh, PA, USA).
9
Comprehensive Antibody Characterization for Molecular Biology
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Antibodies used in this study include αm6A (Millipore, ABE572 or MABE1006) for MeRIP/m6A-seq and MeRIP-qPCR, αHMBOX1 (Novus Biologicals, NBP1-31316) for ChIP-qPCR and immunoblotting, αγH2AX (JBW301, Millipore, 05-636) for immunofluorescence-FISH and Western blot, αTPP1 (Proteintech, 25849-1-AP) for immunoprecipitation (IP) and immunoblotting, and αTRF2 (Proteintech, 66893-1-Ig) for immunofluorescence-FISH. Other antibodies that were applied in Western blot analysis include αMETTL3 (Proteintech, 15073-1-AP), αMETTL14 (Sigma-Aldrich, HPA038002), αALKBH5 (Sigma-Aldrich, HPA007196), αHA (16B12) (BioLegend, #901501), αmyc (9E10) (Santa Cruz Biotechnology, sc-40), αHis (Proteintech, #66005-1), αCRISPR-Cas9 (Abcam, ab191468), αAR (H-280) (Santa Cruz Biotechnology, sc-13062), αYTHDF2 (Proteintech, 24744-1-AP), αYTHDC2 (Proteintech, 27779-1-AP), αp53 (DO-1) (Santa Cruz Biotechnology, sc-126), αp21 (Cell Signaling Technology, #2947), αMDM2 (Proteintech, 19058-1-AP), αTelomerase reverse transcriptase (TERT) (Abcam, ab32020), and αβ-actin (Sigma-Aldrich, A5441). Reagents that were used in IP, ChIP, and MeRIP are Protein A/G Plus Agarose (Santa Cruz Biotechnology, sc-2003) and Dynabeads Protein A (Thermo Fisher Scientific, #10006D) or Protein G (Thermo Fisher Scientific, #10007D) immunoprecipitation kit.
10
Western Blot Analysis of Placental Proteins
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Human and mouse placental tissues and human cultured trophoblast cells were lysed in Ripa buffer (50 mM Tris–HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Na‐D.O.C., 0.1% SDS, 1% Triton X‐100) containing protease inhibitors (Sigma), using a tissue homogenizer. Equal amounts of protein were separated on 12% SDS–polyacrylamide gels and transferred to PVDF membranes. Detection was performed with the following primary antibodies: α‐p21 (BD Biosciences), α‐p53, α‐Stat3, α‐p65 (Santa Cruz Biotechnology), α‐p16 (Abcam), α‐DCR2 (Enzo Life Sciences), α‐Smad3,α‐p‐Smad3, α‐p‐Stat3, α‐p‐p65, α‐Mek1/2, α‐p‐Mek1/2 (Cell Signaling), α‐β‐actin (Sigma), and α‐GAPDH (Merck). The detailed list of antibodies is presented in Appendix Table S5 .
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